Lymphocytes have been studied extensively for the treatment of solid tumors and viral infections, as an adjuvant to bone marrow transplantation, and for the treatment of genetic diseases. For example, early immunotherapy studies for the treatment of melanoma and renal cell carcinoma focused on lymphokine-activated killer (LAK) cells or tumor infiltrating lymphocytes (TILs) (Rosenberg, 1985; Rosenberg, 1987; Rosenberg, 1988), cytotoxic T lymphocytes have been used for the treatment of AIDS (Koenig, 1995; Torpey, 1993; Trickett, 1998), and donor lymphocyte infusions are used after allogeneic bone marrow transplant to enhance graft-versus-leukemia effect and to reduce the potential for relapse (Kolb, 1997). Furthermore, genetically modified peripheral blood lymphocytes (PBLs) have been used in clinical trials for the treatment of severe combined immune deficiency caused by adenosine deaminase (ADA) deficiency (Blaese, 1995), and a variety of other lymphocyte-based therapies have been proposed, including transducing lymphocytes with the herpes simplex virus (HSV) thymidine kinase (TK) suicide gene for allogeneic bone marrow transplantation (Bonini, 1997) and the introduction of cytotoxic T lymphocytes (CTLs) that recognize specific melanomal antigens (Kawakami, 1998).
Cryopreservation of cells that have been expanded and manipulated ex vivo is important for the clinical application of cell-based therapies. Cryopreservation facilitates pooling of cells to reach a therapeutic dose, and facilitates safety testing of both the cell product and any agents, such as recombinant viral vectors or liposomal delivery vehicles, used to genetically modify the cells. Furthermore, as the genetic modification and/or expansion of cells for therapeutic use may require days to weeks for completion of the ex vivo culture protocol, cryopreservation facilitates the coordination of cellular therapy with donor care.
In the method most commonly used for the cryopreservation of bone marrow, peripheral blood lymphocytes (PBLs) are resuspended in a cryopreservation medium containing 10% dimethylsulfoxide (DMSO), autologous plasma and Hank's balanced salt solution (Rowley, 1994; Trickett, 1998) and cooled at 1° C./minute. A second method involves freezing lymphocytes in a 20% glycerol solution supplemented with autologous serum (Areman, 1988), resulting in increased cell viability over the standard DMSO protocol. Glycerol-based cryopreservation solutions have also been reported for hematopoietic progenitor cells (U.S. Pat. No. 5,759,764), however, glycerol has lower permeability, which increases the chance for cell loss from osmotic stresses. Although Oliver et al. (WO 97/35472) relate to a combination of arabinogalactan and cell culture media as useful as a cryopreservation medium, the data was obtained from cell lines, not primary cells.
DMSO cryopreservation involves the risk of DMSO-associated toxicity, particularly where cell transfer therapy is involved. For example, Zambelli et al. (1998) evaluated the infusion-related toxicity of transplanted cryopreserved cells and determined that the amount of DMSO present in the graft is related to the grade of toxicity. Davis et al. (1990) found that almost all patients who received cryopreserved autologous cell grafts exhibited dyspnea (83%), decreased heart rates (98%), and transient hypertension (96%), which were attributed to the infusion of DMSO. Oliguric renal failure and second degree heart block were less frequently observed. Similar results were observed in a study by Stroncek et al. (1991), who found that infusion-related reactions, principally nausea and chills, were associated with transplantation of cryopreserved bone marrow. Moreover, in pediatric patients, higher levels of nausea, vomiting, cardiac arrhythmia and hypotension are noted following transplant of cryopreserved bone marrow (Okamoto, 1993). Since most lymphocyte therapies require the infusion of multiple doses of cells on a regular basis, the toxic effects of DMSO can be cumulative.
The development of appropriate solutions is not the only issue in the development of cryopreservation protocols for lymphocytes which are used therapeutically. Recent studies indicate that in vitro culture influences the freezing response of cells. For example, studies of in vitro cultured hematopoietic progenitor cells and lymphocytes indicates that water transport and intracellular ice formation characteristics of the cells is influenced by in vitro culture (Hubel, 1999). Specifically, subzero water transport characteristics of the cells and postthaw viability were influenced by time in culture.
Thus, what is needed is a cryopreservation composition that is non-toxic and useful for achieving the desired viability rates for cryopreservation of cells for cellular therapy.